Interaction Between Defects in Ventilatory and Thermoregulatory

Interaction between defects in ventilatory and thermoregulatorycontrol in mice lacking 5-HT neurons

Matthew R. Hodges1 and George B. Richerson1,2

1 Departments of Neurology and Cellular and Molecular Physiology, Yale University School of Medicine,New Haven, CT 06520

2 Veterans Affairs Medical Center, West Haven, CT 06516

AbstractWe have previously shown that mice with near-complete absence of 5-HT neurons (Lmx1bf/f/p)display a blunted hypercapnic ventilatory response (HCVR) and impaired cold-inducedthermogenesis, but have normal baseline ventilation (V̇E), core body temperature (TCore) and hypoxicventilatory responses (HVR) at warm ambient temperatures (TAmb; 30°C). These results suggest that5-HT neurons are an important site for integration of ventilatory, metabolic and temperature control.To better define this integrative role, we now determine how a moderate cold stress (TAmb of 25°C)influences ventilatory control in adult Lmx1bf/f/p mice. During whole animal plethysmographicrecordings at 25°C, baseline V̇E, metabolic rate (VO2), and TCore of Lmx1bf/f/p mice were reduced(P<0.001) compared to wild type (WT) mice. Additionally, the HCVR was reduced in Lmx1bf/f/p

mice during normoxic (−33.1%) and hyperoxic (−40.9%) hypercapnia. However, V̇E in Lmx1bf/f/p

mice was equal to that in WT mice while breathing 10% CO2, indicating that non-5-HT neurons mayplay a dominant role during extreme hypercapnia. Additionally, ventilation was decreased duringhypoxia in Lmx1bf/f/p mice compared to WT mice at 25°C due to decreased TCore. These data suggestthat a moderate cold stress in Lmx1bf/f/p mice leads to further dysfunction in ventilatory controlresulting from failure to adequately maintain TCore. We conclude that 5-HT neurons contribute tothe hypercapnic ventilatory response under physiologic, more than during extreme levels of CO2,and that mild cold stress further compromises ventilatory control in Lmx1bf/f/p mice as a result ofdefective thermogenesis.

Keywordshypercapnia; hypoxia; control of breathing; serotonin

1. IntroductionThe primary goal of the respiratory control system is to establish a rate of alveolar gas exchangeto match metabolic demand, and as a result ventilation (V̇E) is closely linked to metabolic rate(V ̇O2). However, changing environmental conditions such as ambient temperature (TAmb),O2 availability, or inspired CO2 leads to a shift in homeostatic strategies in an attempt to

Correspondence: Matthew R. Hodges, PhD, Dept. of Neurology, LCI 704, Yale University School of Medicine, New Haven, CT 06520,Email: [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptRespir Physiol Neurobiol. Author manuscript; available in PMC 2009 December 31.

Published in final edited form as:Respir Physiol Neurobiol. 2008 December 31; 164(3): 350–357. doi:10.1016/j.resp.2008.08.003.

NIH

-PA Author Manuscript

NIH


NIH


maintain core body temperature (TCore) and/or blood gases. V̇E and V ̇O2 are close to theirminimum when TAmb is near or within the thermoneutral zone (~30–32°C in mice (Gordon,C. J., 1985)). Lowering TAmb initiates mechanisms aimed at heat conservation and generation,which increases V̇O2 and consequently V̇E. These shifts in ventilation and metabolism alterthe response strategy to respiratory challenges. For example, in rodents the primary responseto hypoxia (and hypercapnia) under warm ambient conditions is a large increase in V̇E, whereasV ̇O2 is minimally affected (Saiki, C. et al., 1996). In contrast, under cool ambient conditionsthe predominant effect of hypoxia is to lower V̇O2 with little or no change in V̇E (Mortola, J.P. et al., 1995). The hypoxia-induced reduction in V̇O2 results in a decrease in TCore, whichcan independently lower the sensitivity of the ventilatory control system depending upon themagnitude of the temperature drop (Maskrey, M., 1990). For example, severe hypothermia(decreasing TCore to ~28°C) lowers both the hypoxic and HCVRs in dogs (althoughinterpretation of these data is complicated by the use of anesthesia) (Natsui, T., 1969). Smallerdecreases in abdominal temperature (from 37°C to 35°C) in conscious rats using an abdominalheat exchanger leads to little or no change in the response to hypercapnia, whereas increasingTCore augments CO2 sensitivity (Maskrey, M., 1990). In contrast, hypoxia reduces ventilationin animals under these conditions (Maskrey, M., 1990), suggesting that modest hypothermiahas greater effects on ventilatory responses to hypoxia than hypercapnia.

The integration of respiratory, metabolic and thermoregulatory demands is critical for properblood gas, metabolic and temperature homeostasis, and the hypothalamus and raphé 5-HTsystem may both represent sites for such integration (Waldrop, T. G. et al., 1986; Hinrichsen,C. F. et al., 1998; Hodges, M. R. et al., 2008b). The preoptic anterior hypothalamus (POAH)contains warm- and cold-sensitive neurons (Griffin, J. D. et al., 1996), and receives afferentinputs from peripheral thermoreceptors (Boulant, J. A. et al., 1974). Additionally, warm-sensitive POAH neurons and lateral hypothalamic hypocretin-producing neurons are alsoCO2 sensitive, and lesioning orexin neurons blunts the HCVR (Deng, B. S. et al., 2007;Williams, R. H. et al., 2007; Wright, C. L. et al., 2007). Similarly, raphé 5-HT neurons respondto central (Nason, M. W., Jr. et al., 2006) and peripheral (Martin-Cora, F. J. et al., 2000) coolingand augment cold-induced thermogenesis. 5-HT neurons also contribute to central respiratorychemoreception, and facilitate respiratory rhythm generation and respiratory motor output(Al-Zubaidy, Z. A. et al., 1996; Pena, F. et al., 2002; Richerson, G. B., 2004; Hodges, M. R.et al., 2008a).

We have previously examined ventilatory and thermoregulatory control in Lmx1bf/f/p mice, inwhich Lmx1b (LIM homeobox transcription factor 1β) is deleted selectively in neurons thatexpress Pet1 (plasmacytoma expressed transcript 1). This leads to complete and specific 5-HTneuron loss and central 5-HT depletion, without affecting other monoamine systems (Zhao, Z.Q. et al., 2006; Zhao, Z. Q. et al., 2007). Lmx1bf/f/p mice exhibit a severely blunted HCVR andcold-induced thermogenesis despite normal baseline V̇E and V ̇O2, and a normal HVR (Hodges,M. R. et al., 2008b). Those ventilatory measurements were performed at TAmb of 30.0 – 30.8°C (thermoneutral) to prevent confounding effects of changes in TCore. However, thetemperature of most animal facilities is maintained near 25°C, which is below thermoneutraltemperature for a mouse. Previous measurements of TCore in Lmx1bf/f/p mice revealed a failureto maintain TCore at TAmb of 4°C and 12°C, but 24-hour TCore measurements in their homecages at an TAmb of 25°C revealed no differences between genotypes (Hodges, M. R. et al.,2008b). Here we examine ventilation at rest, during hypoxia, and during normoxic andhyperoxic hypercapnia in WT and Lmx1bf/f/p mice at an TAmb of 25°C using flow-throughplethysmography and show that the defects in thermoregulatory and respiratory control inducedby 5-HT system dysfunction combine to cause greater deleterious effects on ventilatory controlduring hypoxia. In addition, we challenged WT and Lmx1bf/f/p mice to pathologically high(10%) levels of CO2 to determine how important 5-HT neurons are for the HCVR underextreme conditions. All data collected at an TAmb of 30°C have been reported previously

Hodges and Richerson Page 2

Respir Physiol Neurobiol. Author manuscript; available in PMC 2009 December 31.

NIH


NIH


NIH


(Hodges, M. R. et al., 2008b), but are included here for direct comparison to those obtained at25°C.

2. Methods2.1 Animal model

The generation of Lmx1bf/f/p mice has previously been described (Zhao, Z. Q. et al., 2006). 22female age-matched (6–12 months old, see also Table 1) WT (n = 12) and Lmx1bf/f/p (n = 10)mice were used in this study. WT and Lmx1bf/f/p littermates were paired during testing whenpossible.

2.2 PlethysmographyVentilation and oxygen consumption were measured using standard flow-throughplethysmographic techniques (Drorbaugh, J. E. et al., 1955), as described previously (Hodges,M. R. et al., 2008b). Compressed gas mixtures contained: 21% O2 with 0, 3, 5, 7, or 10%CO2 (normoxic hypercapnia; balance N2), 50% O2 with 0, 3, 5, 7, or 10% CO2 (hyperoxichypercapnia; balance N2), or 10% O2 (hypoxia; balance N2). Hypercapnia studies consistedof >20 minutes of baseline, followed by 10-minute exposures of 3, 5, 7, and 10% CO2, eachinterrupted by 10-minute baseline measurements. Similarly, hypoxia studies consisted of >20minutes of baseline followed by 10 minutes of 10% O2. The plethysmograph was set on topof a telemeter energizer/receiver (Model ER-4000, Mini Mitter, Bend, OR) for continuousmeasurement of core body temperature. Air temperature (25–26 °C) and humidity (OmegaHX-93AV, Omega Engineering Inc., Stamford, CT), animal temperature, breathing-inducedpressure oscillations (DC002NDR5, Honeywell International, Morristown, NJ), and O2 andCO2 concentrations (Models CD-3A (CO2) and S3A/I (O2), AEI Technologies Inc., Naperville,IL) were measured continuously, sampled at 100 Hz, digitized using an A/D converter(PCI-6221, National Instruments, Austin, TX) and monitored/stored on disk using a custom-written data acquisition program (Matlab, The MathWorks, Natick, MA). The outflow gasessampled for O2 and CO2 concentrations were dried using a dessication column and measuredwith continuous flow (200 ml/min) through the analyzers. Oxygen consumption was calculatedby subtracting the outflow fraction of O2 from the inflow fraction of O2, and multiplying thedifference by the chamber flow rate (700 ml/min) measured using a flow meter.

2.3 Telemetry Probe ImplantationThe methods for implantation of telemetric temperature probes have been published (Hodges,M. R. et al., 2008b). Briefly, mice were given pre-operative analgesia (meloxicam (1.0 mg/kgI.P.) or buprenorphine (0.1 mg/kg I.P.)) prior to induction and maintenance of anesthesia with20% (v/v) isoflurane mixed with polyethylene glycol. Telemetric temperature probes (EmitterG2, Minimitter, Bend, OR) were implanted into the abdomen using a ventral midline incision,and the wound was sutured. Mice received 1.7 μg/ml Meloxicam for 2 days, and studied > 7days post-op.

2.4 Data analysisAll data were analyzed off-line using custom-written software by an individual blind to theanimal genotypes. All samples of continuous ventilatory data segments of 6–10 secondduration that did not contain sighs, coughs, sniffing or movement artifacts were selected foranalysis during the last five-minute period of exposure to each gas mixture for the normoxicand hyperoxic hypercapnia studies. The average number of 6–10 second segments analyzedunder control conditions was 35.0 ± 3.3 and 34.9 ± 2.3 for WT and (Lmx1bf/f/p) mice,respectively, roughly corresponding to 600–1000 breaths analyzed per animal during thecontrol period. Hypoxia data were analyzed during minutes 2–10 of the 10-minute exposure,



NIH


NIH


NIH


and divided into 2-minute segments to evaluate the time-course of the response. Inspiratorytime (TI, seconds), expiratory time (TE, seconds), inter-breath interval (IBI, seconds, used tocalculate respiratory frequency (fR), breaths·minute−1), standard deviation of IBI (seconds),tidal volume (VT, μl), oxygen consumption (V̇O2, ml·min−1) and minute ventilation (V̇E,ml·min−1, which is the product of VT and fR), were calculated for all animals under allconditions, with the exception of V̇O2 during hyperoxia due to an inability to accuratelymeasure high (>48%) O2 concentrations. VT, V̇E and V̇O2 were normalized to animal weight.

2.5 StatisticsAll data are presented as mean ± SEM. Comparisons were made using a two-way ANOVA(SYSTAT 11, Systat Software, Inc., San Jose, CA) and valid pair-wise comparisons usingeither a paired t-test or t-test assuming unequal variances (Excel, Microsoft Corp.), whenappropriate. The threshold for significance was P < 0.05.

All animals were housed and maintained in the Yale Animal Resource Center and all protocolsapproved by the Yale Animal Care and Use Committee.

3. Results3.1 Baseline ventilation and metabolic rates are reduced in Lmx1bf/f/p mice

Lmx1bf/f/p mice had significantly reduced mass compared to their WT littermates at the agesstudied (Table 1). Therefore, all variables that would be affected by weight differences, suchas minute ventilation (V̇E), tidal volume (VT), and oxygen consumption (V̇O2) were normalizedto body weight. In contrast to previous studies where TAmb was held between 30.0 – 30.8°C,in the current study we made all measurements at an TAmb of 25°C. Under these conditions,VT was equal in WT and Lmx1bf/f/p mice, but V̇E was reduced in Lmx1bf/f/p mice due to a lowerbreathing frequency at rest (Table 1, Fig. 1A & C). The lower frequency was due to an increasedinspiratory time (TI), with expiratory time (TE) equal between genotypes. As a result of thelonger TI, ventilatory drive (VT/TI) was significantly reduced in Lmx1bf/f/p mice. In addition,both V̇O2 and TCore were significantly lower in Lmx1bf/f/p mice relative to WT mice at rest.Therefore, the decrease in V̇E was largely due to the decreased metabolic demand. The V̇E/V ̇O2 ratio was equal at baseline in WT and Lmx1bf/f/p mice (P = 0.18).

3.2 Ventilatory responses to hypoxia and hypercapnia are blunted in Lmx1bf/f/p miceWe challenged WT and Lmx1bf/f/p mice to hypoxia and both normoxic (FIO2= 0.21) andhyperoxic (FIO2 = 0.50) hypercapnia at TAmb of 25°C to determine the effects of a moderatecold stress on the response to these challenges.

3.2.1 Normoxic hypercapniaIn normoxia, we found significant effects of both genotype (P ≤ 0.001) and condition (P ≤0.001) on V ̇E and body temperature, and condition, fR, TI, TE, VT/TI effects on VT (P < 0.0001;two-way ANOVA; Fig. 1). Specifically, V̇E was reduced in Lmx1bf/f/p mice compared to WTmice when breathing room air, 3, 5 and 7% CO2 (Fig. 1A). However, there was no differencein V̇E between genotypes breathing 10% CO2 in normoxia (P = 0.39). We also compared theincrease in V̇E at each level of inspired CO2 relative to the baseline (Fig. 1B), and found thatthe increase in V̇E was significantly reduced breathing 3% and 5% CO2 in Lmx1bf/f/p comparedto WT mice. The blunted HCVR was due solely to a smaller frequency response inLmx1bf/f/p mice (Fig. 1C), with VT increasing equally in WT and Lmx1bf/f/p mice (Fig. 1D).There was also a longer TI in Lmx1bf/f/p mice (P < 0.002), and consequently a smaller VT/TIduring all CO2 levels in normoxia (P ≤ 0.002; data not shown). TCore was significantly reducedin Lmx1bf/f/p (36.2 ± 0.2°C) relative to WT (37.7 ± 0.1°C) mice at baseline during normoxia,



NIH


NIH


NIH


and remained unchanged from baseline in both genotypes while breathing 3, 5 & 7% CO2 (Fig.2E). However, TCore dropped significantly relative to baseline in both genotypes whenbreathing 10% CO2, likely due to increased evaporative and convective heat loss due to thehyperpnea. Unlike WT mice, TCore in Lmx1bf/f/p mice was significantly lower at an TAmb of25°C relative to 30°C during plethysmographic recordings (Fig. 1E). The V̇E/V̇O2 ratio inLmx1bf/f/p mice was equal to WT mice while breathing 3% (P = 0.09), 5% (P = 0.1) and 7%(P = 0.085) CO2 due to reductions in both V̇E and V ̇O2 (Fig. 1F). However, it is important tonote that this measure was significantly reduced in Lmx1bf/f/p mice relative to WT mice whenmeasured at an TAmb of 30°C (Hodges, M. R. et al., 2008b).

3.2.2 Hyperoxic hypercapniaDuring hyperoxia, there were significant effects of both genotype (P < 0.01) and condition (P≤ 0.03) on V ̇E, fR, TI, TE, VT/TI and body temperature, and condition effects on VT (P < 0.0001;two-way ANOVA; Fig. 2). Hyperoxia decreased baseline V̇E in WT mice from 1.32 ± 0.06 ml· min−1 · g−1 (normoxia) to 1.15 ± 0.06 ml · min−1 · g−1 (hyperoxia: P = 0.015), but not inLmx1bf/f/p mice, where V̇E in normoxia was 1.04 ± 0.06 ml · min−1 · g−1 and 1.05 ± 0.04 ml ·min−1 · g−1 in hyperoxia (P = 0.907). However, hyperoxia per se had no effect on V̇E duringhypercapnia relative to normoxia in both WT and Lmx1bf/f/p mice (P > 0.05, two-wayANOVA). Lmx1bf/f/p mice had a lower V̇E. than WT mice while breathing 3, 5, and 7% CO2,but V̇E was not different when breathing room air or 10% CO2 (Fig. 2A). The change in V̇Erelative to baseline was also reduced in Lmx1bf/f/p mice breathing 3, 5, and 7% CO2, but not10% CO2 (Fig. 2B). Lmx1bf/f/p mice also had a lower breathing frequency than WT mice underall conditions in hyperoxia, with no differences in VT (Fig. 2C & D). Additionally, TI wasgreater (P ≤ 0.02) and VT/TI less (P ≤ 0.0006) in Lmx1bf/f/p mice relative to WT mice at restand at all CO2 levels tested in hyperoxia (data not shown). Similar to results in normoxia,TCore was significantly lower in Lmx1bf/f/p mice (36.4 ± 0.1°C) relative to WT (37.8 ± 0.1°C)mice at baseline during hyperoxia (Fig. 2E). TCore also dropped significantly from baselinewhen breathing 10% CO2 in both genotypes. However, there were no differences in TCorebetween genotypes at an TAmb of 30°C (Fig. 2E).

3.2.3 HypoxiaIn contrast to our previous experiments in which TAmb was 30°C (Hodges, M. R. et al.,2008b), there were several differences in the response of Lmx1bf/f/p and WT mice to hypoxiawhen TAmb was 25°C (Fig. 3). There were significant effects of both genotype and conditionon V̇E, fR, TI, TE, and body temperature, and there were genotype effects on VT/TI (P < 0.05;ANOVA). Specifically, V̇E (Fig. 3A), fR (Fig. 3B), V ̇E/V̇O2 (Fig. 3D) and VT/TI were reducedthroughout minutes 2 – 10 of the hypoxic challenge in Lmx1bf/f/p mice, with no effects onVT (Fig. 3C). Interestingly, we did not observe a significant decrease in V̇E or V ̇O2 inLmx1bf/f/p mice relative to WT mice during the control period before the hypoxia challengesas seen prior to the hypercapnia challenges, despite a similar difference in TCore. This may berelated to variability in the time required for some animals to shift from an exploratory, activebehavior to quiet wakefulness. However, both WT and Lmx1bf/f/p mice significantly decreasedV ̇O2 during hypoxia, with no difference between genotypes (Fig. 3E). This result indicates thatthe lower V̇E/V̇O2 during hypoxia was due to a larger reduction in V̇E than in V̇O2. TCore inboth WT and Lmx1bf/f/p mice significantly decreased over time during hypoxia, but the initialtemperature was lower in Lmx1bf/f/p mice compared to WT mice (Fig. 3F).

We then compared these data to those obtained from WT and Lmx1bf/f/p mice studied at anTAmb of 30°C (Hodges, M. R. et al., 2008b). For comparison, we normalized the data obtainedduring hypoxia at both ambient temperatures to the control period, and expressed it as % ofcontrol. We found no differences in V̇E (% control; Fig. 4A) or Δ V̇O2 (relative to room airbreathing; Fig. 4B) between WT and Lmx1bf/f/p mice during hypoxia studied at an TAmb of



NIH


NIH


NIH


either 25 or 30°C. However, TAmb had significant effects on the HVR. At 25°C both genotypesresponded to hypoxia with a small increase in V̇E and large decrease in V̇O2. In contrast, at 30°C they responded to hypoxia with a large increase in V̇E and small decrease in V̇ O2. We alsofound no difference in V̇E/V̇O2 (% control) between WT and Lmx1bf/f/p mice during hypoxiaat 30°C, but V̇E/V̇O2 was lower in Lmx1bf/f/p mice compared to WT mice during hypoxia at25°C due to the smaller increase in V̇E(Fig. 4C). TCore was not different between WT andLmx1bf/f/p mice studied at 30°C while breathing room air and during hypoxia, and TCore didnot change in either genotype in response to hypoxia (Fig. 4D). In contrast, WT andLmx1bf/f/p mice studied at an TAmb of 25°C both had a lower TCore compared to an TAmb of30°C, and at the lower TAmb there was a significant decrease in TCore over time in response tohypoxia.

4. DiscussionMice in which CNS 5-HT neurons have been genetically deleted (Lmx1bf/f/p: (Zhao, Z. Q. etal., 2006) have previously been characterized, and have severe deficits in the HCVR and cold-induced thermogenesis (Hodges, M. R. et al., 2008b). When studied at an TAmb of 30°C, thesemice have normal baseline V̇E, V ̇O2, TCore and HVR. We show here that when studied undermild cold stress (TAmb of 25°C combined with convective heat loss due to airflow in theplethysmograph), Lmx1bf/f/p mice continue to display an attenuated HCVR, but now also havedecreased baseline V̇E, V ̇O2, TCore, and ventilatory response to hypoxia. These results areconsistent with the conclusion that the primary defects in Lmx1bf/f/p mice are a reduced HCVRand thermogenic capabilities, and that the decreased baseline ventilation and HVR aresecondary to dysfunctional thermogenesis. Thus, a mild cold stress further compromisesventilatory control as a consequence of the thermoregulatory deficit in mice with 5-HT systemdysfunction.

4.1 Methodological considerationsMammals respond to a decreased TAmb by initiating mechanisms that drive heat conservationand generation, which increases V̇O2 and consequently ventilation (Mortola, J. P., 2005).Indeed, both WT and Lmx1bf/f/p mice had increased baseline ventilation and V̇O2 underconditions of mild cold stress relative to measurements at 30°C, but V̇O2 and TCore were lowerin Lmx1bf/f/p mice compared to WT mice in these cooler conditions. This indicates that 25°Cis below the lower critical temperature for these mice, and is consistent with our previousobservations of thermoregulatory dysfunction (Hodges, M. R. et al., 2008b). However, wepreviously found no difference in TCore in WT and Lmx1bf/f/p mice measured during 24-hourrecordings in their home cages at 25°C. This indicates that a mildly decreased TAmb is notsufficient alone to decrease TCore in Lmx1bf/f/p mice. It is therefore important to consider thedesign of the plethysmographic measurements.

We used a flow through plethysmographic chamber with a flow rate of 700 ml·min−1 to ensurerapid gas changes while retaining a large enough drop in O2 concentration to allow accurateV ̇O2 measurements. This high flow rate would be expected to cause substantial convective heatloss, and a greater thermal challenge than would occur by decreasing TAmb alone. Theattenuated thermogenic response to cold would lead to an exaggerated drop in TCore inLmx1bf/f/p mice. In addition, the small dimensions of the plethysmograph chamber allowed forfree, but somewhat restricted movement, which itself can affect TCore and V ̇O2 (Cinelli, P. etal., 2007). This could influence TCore in a variety of ways, including increasing heat generationdue to stress-induced activation of the hypothalamic-pituitary-adrenal axis and catecholaminerelease (Harris, R. B. et al., 2002), while decreasing heat generation due to reduced motoractivity. The convective airflow combined with lower TAmb, restricted movement, and the



NIH


NIH


NIH


known thermogenic defects in Lmx1bf/f/p mice likely contributed to a significantly lowerTCore.

4.2 Primary and secondary effects of an absence of 5-HT neuronsIn our previous work (Hodges, M. R. et al., 2008b) we found that the primary effect of near-complete absence of 5-HT neurons was a blunted hypercapnic response and impaired cold-induced thermogenesis. In contrast, there was no effect on baseline V̇E, V̇O2 and TCore. Here,we show that under cool conditions Lmx1bf/f/p mice display reduced V̇E, V ̇O2 and TCore atbaseline, and decreased V̇E/V̇O2 ratio during hypoxia. We conclude that the lower ventilationat rest and during hypoxia under conditions of moderate cold stress is due to the decreasedTCore in Lmx1bf/f/p mice, which is secondary to a primary defect in heat generation (Hodges,M. R. et al., 2008b). While the concept of decreased body temperature blunting the ventilatoryresponse to hypoxia is not novel, these data suggest that when the 5-HT system is not workingnormally a modest thermal challenge has secondary deleterious effects on ventilatory controlas a result of the primary defect in thermogenesis.

Proper coupling of ventilation and metabolism is particularly important during a hypoxicchallenge, where the integrated response normally includes both hyperpnea andhypometabolism. At an TAmb near the thermoneutral range, WT and Lmx1bf/f/p mice respondto hypoxia with a robust hyperpnea and only a small decrease in metabolism, whereas whenTAmb is cooler both genotypes exhibited only a small increase in ventilation and a largedecrease in V̇O2. In addition, both genotypes maintained a constant TCore during hypoxia at30°C, but TCore decreased significantly in both genotypes during hypoxia at 25°C. Thissuggests that Lmx1bf/f/p mice retain the ability to shift strategies in changing ambientconditions, but are clearly less effective in maintaining TCore. In addition, the data suggest that5-HT neurons do not contribute directly to the HVR or hypoxia-induced hypothermia per se,suggesting that the mechanism by which hypoxia inhibits V̇O2 (and subsequently decreasesTCore) is independent of raphé 5-HT neurons.

4.3 Effects on the hypercapnic ventilatory responseAt an TAmb of 30°C, the HCVR of Lmx1bf/f/p mice is reduced by 42.2% in normoxia and by51.6% in hyperoxia (Hodges, M. R. et al., 2008b). Under those conditions, there were nodifferences between Lmx1bf/f/p and WT mice in V̇O2 or TCore at baseline or during hypercapnia,indicating that the deficit in the HCVR is a direct result of the absence of 5-HT and/or 5-HTneurons, and not an indirect effect from altered thermoregulation. Here we found thatventilation while breathing 3, 5 and 7% inspired CO2 was reduced by 33.1% in normoxia andby 40.9% in hyperoxia at an TAmb of 25°C, slightly less but similar to our previous findings.This result is similar to previous reports showing a greater effect of mild hypothermia on theHVR compared to the HCVR (Maskrey, M., 1990).

We previously found that relative to wild type mice, Lmx1bf/f/p mice have a significantlysmaller increase in ventilation when challenged with 5% and 7% CO2, but there was nodifference in ventilation at baseline or in response to 3% CO2 at 30°C (Hodges, M. R. et al.,2008b). Here we found that the ventilatory response to 3%, 5% and 7% CO2 was less inLmx1bf/f/p than WT mice at 25°C. However, there was no difference in ventilation betweengenotypes breathing 10% CO2 in both normoxia and hyperoxia at 25°C. These data supportthe hypothesis that 5-HT neurons make their greatest contribution to central chemoreceptionat low levels of CO2 that would occur under physiological conditions, consistent with theirhigh degree of intrinsic chemosensitivity to small changes in pH in vitro (Richerson, G. B.,1995; Wang, W. et al., 2001; Bradley, S. R. et al., 2002; Wang, W. et al., 2002; Richerson, G.B., 2004). The data are also consistent with the hypothesis that non-5-HT neurons make theirgreatest contributions under conditions of severe hypercapnia (Nattie, E., 1999).



NIH


NIH


NIH


In contrast to our previous findings at a TAmb of 30°C, the decreased ventilation duringhypercapnia was not accompanied by a significant reduction in the V̇E/V̇O2 ratio, indicatingthat under moderate cold stress Lmx1bf/f/p mice retain the ability to generate hyperpneaproportional to metabolism during hypercapnia. The reason that different results were obtainedat the two temperatures is unclear, but there are several considerations. One possibility is thata subset of non-5-HT chemoreceptors (including peripheral chemoreceptors) play a greaterrole at colder temperature as a result of an interaction between temperature regulation andchemoreception. Another possibility is that cold exposure leads to an increase in tonicrespiratory drive from non-5-HT sources, which could mask the established primary deficit inV ̇E relative to metabolism (Hodges, M. R. et al., 2008b). A third is that 5-HT neurons mediatea decrease in oxygen consumption in response to hypercapnia during cold exposure but notunder thermoneutral conditions. Finally, it is important to note that it is difficult to accuratelymeasure O2 consumption in a small species such as a mouse, and even small errors inmeasurement of V̇O2 could lead to inability to detect real differences. The p values comparingthese data were close to the threshold for significance, preventing us from ruling out a small,but real deficit in the V̇E/V̇O2 ratio of Lmx1bf/f/p mice. However, the existing data suggest thatthere may be a greater degree of compensation for the loss of 5-HT neurons under thermalstress relative to thermoneutral conditions. The source of this compensation is unclear, but itmay include an increase in the contribution of peripheral or other central chemoreceptors.

Consistent with developmental compensation, hyperoxia decreased resting ventilation in WTmice relative to normoxia, but had no effect on resting ventilation in Lmx1bf/f/p mice. Thissuggests that there may be altered carotid body function or central processing of carotid bodyinput in response to the loss of 5-HT neurons. If there is compensation by peripheralchemoreceptors (or possibly by non-5-HT central chemoreceptors) this would lead to anunderestimation of the role of 5-HT neurons in the HCVR. As discussed previously (Hodgeset al, 2008), this role of 5-HT neurons in the HCVR likely includes both a direct role in sensingCO2 and an indirect role in enhancing chemosensitivity of non-5-HT neurons.

4.4 Summary and ConclusionsVentilation and TCore can be regulated normally in Lmx1bf/f/p mice under conditions of minimalenvironmental stress, but both become abnormal when challenged. This is consistent with theidea that 5-HT neurons play a major role in respiratory and thermoregulatory homeostasis underconditions of environmental stress. Abnormalities in the 5-HT system have been identified insudden infant death syndrome (SIDS) (Paterson, D. S. et al., 2006), which is thought to be dueto the inability of a vulnerable infant to maintain homeostasis when challenged by an exogenousstressor (Kinney, H. C., 2005). Based on these new, and previous (Richerson, G. B., 2004;Hodges, M. R. et al., 2008b) data, we conclude that: 1) 5-HT neurons contribute significantlyto the HCVR and cold-induced thermogenesis; 2) 5-HT neurons participate in the integrationof ventilatory and metabolic demands, and; 3) A mild thermal stress can lead to worsening ofthe deficits in ventilatory control that are caused by an abnormal 5-HT system.

AcknowledgementsWe thank Drs. E.E. Nattie, R.A. Darnall, H.C. Kinney, J.C. Leiter, D. Bartlett, J.A. Daubenspeck and S.M. Dymeckifor their insights and thoughtful discussions. We also thank Joe Murillo, Yin Jun, Elisa Yin and Dr. Zhou-Feng Chenfor animal genotyping, protocols and primers. This work was supported by the Parker B. Francis Foundation (M.R.H.),NIH P01HD36379, NIH HD052772, and the VAMC (G.B.R.).



NIH


NIH


NIH


Reference ListAl-Zubaidy ZA, Erickson RL, Greer JJ. Serotonergic and noradrenergic effects on respiratory neural

discharge in the medullary slice preparation of neonatal rats. Pflugers Arch 1996;431:942–949.[PubMed: 8927513]

Boulant JA, Hardy JD. The effect of spinal and skin temperatures on the firing rate and thermosensitivityof preoptic neurones. J Physiol 1974;240:639–660. [PubMed: 4416218]

Bradley SR, Pieribone VA, Wang W, Severson CA, Jacobs RA, Richerson GB. Chemosensitiveserotonergic neurons are closely associated with large medullary arteries. Nat Neurosci 2002;5:401–402. [PubMed: 11967547]

Cinelli P, Rettich A, Seifert B, Burki K, Arras M. Comparative analysis and physiological impact ofdifferent tissue biopsy methodologies used for the genotyping of laboratory mice. Lab Anim2007;41:174–184. [PubMed: 17430617]

Deng BS, Nakamura A, Zhang W, Yanagisawa M, Fukuda Y, Kuwaki T. Contribution of orexin inhypercapnic chemoreflex: Evidence from genetic and pharmacological disruption andsupplementation studies in mice. J Appl Physiol. 2007

Drorbaugh JE, FENN WO. A barometric method for measuring ventilation in newborn infants. Pediatrics1955;16:81–87. [PubMed: 14394741]

Gordon CJ. Relationship between autonomic and behavioral thermoregulation in the mouse. PhysiolBehav 1985;34:687–690. [PubMed: 4034706]

Griffin JD, Kaple ML, Chow AR, Boulant JA. Cellular mechanisms for neuronal thermosensitivity inthe rat hypothalamus. J Physiol 1996;492(Pt 1):231–242. [PubMed: 8730598]

Harris RB, Mitchell TD, Simpson J, Redmann SM Jr, Youngblood BD, Ryan DH. Weight loss in ratsexposed to repeated acute restraint stress is independent of energy or leptin status. Am J Physiol RegulIntegr Comp Physiol 2002;282:R77–R88. [PubMed: 11742826]

Hinrichsen CF, Maskrey M, Mortola JP. Ventilatory and metabolic responses to cold and hypoxia inconscious rats with discrete hypothalamic lesions. Respir Physiol 1998;111:247–256. [PubMed:9628230]

Hodges MR, Richerson GB. Contributions of 5-HT neurons to respiratory control: Neuromodulatory andtrophic effects. Respir Physiol Neurobiol. 2008a

Hodges MR, Tattersall G, Harris MB, McEvoy S, Richerson D, Deneris ES, Johnson RL, Chen ZF,Richerson GB. Defects in breathing and thermoregulation in mice with near-complete absence ofcentral serotonin neurons. J Neurosci 2008b;28:2495–2505. [PubMed: 18322094]

Kinney HC. Abnormalities of the brainstem serotonergic system in the sudden infant death syndrome: areview. Pediatr Dev Pathol 2005;8:507–524. [PubMed: 16222475]

Martin-Cora FJ, Fornal CA, Metzler CW, Jacobs BL. Single-unit responses of serotonergic medullaryand pontine raphe neurons to environmental cooling in freely moving cats. Neuroscience2000;98:301–309. [PubMed: 10854761]

Maskrey M. Body temperature effects on hypoxic and hypercapnic responses in awake rats. Am J Physiol1990;259:R492–R498. [PubMed: 2118731]

Mortola JP. Influence of temperature on metabolism and breathing during mammalian ontogenesis.Respir Physiol Neurobiol 2005;149:155–164. [PubMed: 16126013]

Mortola, JP.; Gautier, H. Interaction between metabolism and ventilation: effects of respiratory gasesand temperature. In: Dempsey, JA.; Pack, AI., editors. Regulation of Breathing. Marcel Dekker; NewYork: 1995. p. 1011-1064.

Nason MW Jr, Mason P. Medullary raphe neurons facilitate brown adipose tissue activation. J Neurosci2006;26:1190–1198. [PubMed: 16436606]

Natsui T. Respiratory response to hypoxia with hypocapnia or normocapnia and to CO2 in hypothermicdogs. Respir Physiol 1969;7:188–202. [PubMed: 5823832]

Nattie E. CO2, brainstem chemoreceptors and breathing. Prog Neurobiol 1999;59:299–331. [PubMed:10501632]

Paterson DS, Trachtenberg FL, Thompson EG, Belliveau RA, Beggs AH, Darnall R, Chadwick AE,Krous HF, Kinney HC. Multiple serotonergic brainstem abnormalities in sudden infant deathsyndrome. JAMA 2006;296:2124–2132. [PubMed: 17077377]



NIH


NIH


NIH


Pena F, Ramirez JM. Endogenous activation of serotonin-2A receptors is required for respiratory rhythmgeneration in vitro. J Neurosci 2002;22:11055–11064. [PubMed: 12486201]

Richerson GB. Serotonergic neurons as carbon dioxide sensors that maintain pH homeostasis. Nat RevNeurosci 2004;5:449–461. [PubMed: 15152195]

Richerson GB. Response to CO2 of neurons in the rostral ventral medulla in vitro. J Neurophysiol1995;73:933–944. [PubMed: 7608778]

Saiki C, Mortola JP. Effect of CO2 on the metabolic and ventilatory responses to ambient temperaturein conscious adult and newborn rats. J Physiol 1996;491(Pt 1):261–269. [PubMed: 9011618]

Waldrop TG, Mullins DC, Millhorn DE. Control of respiration by the hypothalamus and by feedbackfrom contracting muscles in cats. Respir Physiol 1986;64:317–328. [PubMed: 3738257]

Wang W, Bradley SR, Richerson GB. Quantification of the response of rat medullary raphe neurones toindependent changes in pH(o) and P(CO2). J Physiol 2002;540:951–970. [PubMed: 11986382]

Wang W, Tiwari JK, Bradley SR, Zaykin RV, Richerson GB. Acidosis-stimulated neurons of themedullary raphe are serotonergic. J Neurophysiol 2001;85:2224–2235. [PubMed: 11353037]

Williams RH, Jensen LT, Verkhratsky A, Fugger L, Burdakov D. Control of hypothalamic orexin neuronsby acid and CO2. Proc Natl Acad Sci U S A 2007;104:10685–10690. [PubMed: 17563364]

Wright CL, Boulant JA. Carbon dioxide and pH effects on temperature-sensitive and -insensitivehypothalamic neurons. J Appl Physiol 2007;102:1357–1366. [PubMed: 17138840]

Zhao ZQ, Chiechio S, Sun YG, Zhang KH, Zhao CS, Scott M, Johnson RL, Deneris ES, Renner KJ,Gereau RW, Chen ZF. Mice lacking central serotonergic neurons show enhanced inflammatory painand an impaired analgesic response to antidepressant drugs. J Neurosci 2007;27:6045–6053.[PubMed: 17537976]

Zhao ZQ, Scott M, Chiechio S, Wang JS, Renner KJ, Gereau RW, Johnson RL, Deneris ES, Chen ZF.Lmx1b is required for maintenance of central serotonergic neurons and mice lacking centralserotonergic system exhibit normal locomotor activity. J Neurosci 2006;26:12781–12788. [PubMed:17151281]



NIH


NIH


NIH


Figure 1. The normoxic hypercapnic ventilatory response and body temperature are reduced inLmx1bf/f/p mice at 25°CA, Minute ventilation (V̇E), B, change in ventilation from baseline, C, respiratory frequency(fR), D, tidal volume (VT), E, core temperature (TCore) and F, oxygen consumption (V̇O2) atrest and breathing 0%, 3%, 5%, 7% and 10% inspired CO2 in normoxia (FIO2 = 0.21) at roomtemperature (RT: 25°C; A–D, F) or thermoneutral (TN: 30°C (Hodges, M. R. et al., 2008b))in WT (n=12) and Lmx1bf/f/p mice (n=10). Two-way ANOVA (genotype and condition, orambient temperature as factors) or unpaired t-test, * denotes P<0.05 for WT versusLmx1bf/f/p, # denotes P<0.05 for 10% CO2 versus baseline. Data are mean ± SEM.



NIH


NIH


NIH


Figure 2. The hyperoxic hypercapnic ventilatory response and body temperature are reduced inLmx1bf/f/p mice at 25°CA, Minute ventilation (V̇E), B, change in ventilation from baseline, C, respiratory frequency(fR), D, tidal volume (VT), and E, core temperature (TCore) at rest and breathing 0%, 3%, 5%,7% and 10% inspired CO2 in hyperoxia (FIO2 = 0.5) at room temperature (RT: 25°C) orthermoneutral (TN: 30°C (Hodges, M. R. et al., 2008b)) in WT (n=12) and Lmx1bf/f/p mice(n=10). Two-way ANOVA (genotype and condition, or ambient temperature as factors) orunpaired t-test, * denotes P<0.05 for WT versus Lmx1bf/f/p, # denotes P<0.05 for 10% CO2versus baseline. Data are mean ± SEM.



NIH


NIH


NIH


Figure 3. Lmx1bf/f/p mice have a blunted hypoxic ventilatory response at 25°CA, Minute ventilation (V̇E), B, respiratory frequency (fR), C, tidal volume (VT), D, V ̇E/V̇O2ratio, E, V̇O2, and F, core temperature (TCore) breathing room air (RA) and during minutes 2–10 of a 10-minute hypoxia challenge (FIO2 = 0.1) in WT (n=9) and Lmx1bf/f/p mice (n=7). Two-way ANOVA (genotype and time as factors) and unpaired t-test, * denotes P<0.05 for WTversus Lmx1bf/f/p mice. Data are mean ± SEM.



NIH


NIH


NIH


Figure 4. The ventilation to oxygen consumption ratio is reduced at 25°C due to a reduced bodytemperature in Lmx1bf/f/p miceA, Minute ventilation (VE; % control), B, V̇O2, C, V ̇E/VO2 ratio (% control), and D, coretemperature (TCore) in WT (solid symbols) and Lmx1bf/f/p (open symbols) mice at TAmb of 25°C (squares) and 30°C (triangles (Hodges, M. R. et al., 2008b)). Note that both genotypes shiftventilatory and metabolic strategies during hypoxia at different TAmb, and that the V̇E/VO2ratio is reduced under cool conditions. Two-way ANOVA (genotype and time, or TAmb asfactors) and unpaired t-test, * denotes P<0.05 for WT versus Lmx1bf/f/p mice, # denotes P<0.05for 25°C versus 30°C. Data are mean ± SEM.



NIH


NIH


NIH


NIH


NIH


NIH



Table 1Baseline parameters in WT and Lmx1bf/f/p mice

Parameter Wild Type Lmx1bf/f/p

Age (days) 263.6 ± 9.9 264.6 ± 17.5Weight (gm) 33.0 ± 1.6 28.5 ± 1.1 *TCore (°C) 37.9 ± 0.2 36.4 ± 0.2 **V ̇E (ml · min−1 gm−1) 1.32 ± 0.06 1.04 ± 0.06 **fR (breaths · min−1) 172.9 ± 6.9 143.6 ± 4.7 **VT (μl · breath−1 · gm−1) 7.71 ± 0.37 7.24 ± 0.33TI(sec−1) 0.1 ± 0.004 0.175 ± 0.007 **TE (sec−1) 0.255 ± 0.012 0.255 ± 0.009VT/TI(ml · breath−1·sec−1) 2.55 ± 0.14 1.20 ± 0.08 **V ̇O2 (ml O2 min−1·gm−1) 0.066 ± 0.003 0.056 ± 0.003 *V ̇E/V̇O2 20.8 ± 1.3 19.0 ± 0.7

All values are mean (± SEM); Age (at the time of study), core temperature (TCore), minute ventilation (V̇E), frequency (fR), tidal volume (VT), inspiratory

time (TI), expiratory time (TE), ventilatory drive (VT/TI), oxygen consumption (V̇O2), convection requirement (V̇E/V̇O2). All measurements were

obtained while breathing room air at an ambient temperature of 25°C. Comparisons were made with an unpaired t-test.

*denotes P<0.05,

**denotes P<0.005.


Date post:	10-Apr-2016
Category:	Documents
Upload:	rehaan-fayaz
View:	220 times
Download:	1 times

Interaction Between Defects in Ventilatory and Thermoregulatory

Documents